The overall objective of the proposed research is to elucidate the mechanisms by which cartilage, muscle and other connective tissues differentiate in their proper patterns within the embryonic limb bud. Monoclonal antibodies directed against antigens which are specific to early connective tissue percursor cells or their associated extracellular matrix will be prepared. The antigens of interest will be used as markers to follow the origin and fate of the precursor cells in situ by use of immunohistochemistry. In addition, attention will be focused on antibodies that can disrupt normal differentiation in high cell density cultures of limb mesenchyme. Such antigens will be characterized and isolated by antibody affinity chromatography. Also, efforts will be made to determine the roles of interactions between mesenchyme cells and between epithelia and mesenchyme in the origin of cell diversity in the limb bud. Previous work in this laboratory has shown that young limb mesenchyme cells produce a chondrogenic inducer. Studies are proposed concerning the nature of the inducer, its interaction with fibronectin and the relationships between the inducer, the cytoskeleton and endogenous levels of cyclic AMP. Previous results from this laboratory indicate that through a diffusible factor the limb ectoderm can influence whether cartilage or soft connective tissue differentiates. Efforts will be made to study the effects of the diffusible factor by use of ectoderm-conditioned hydrated type I collagen gels as a culture milieu. The effects of the ectoderm on cell shape and cyclic nucleotide levels will be examined. In addition, monoclonal antibody techniques will be used to characterize epithelially derived factors that influence mesenchymal cell differentiation.